Recent advances in understanding the metabolic origin and the temporal dynamics in δ13C of dark-respired CO2 (δ13Cres) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in δ13Cres have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in δ13C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in δ13Cres (ranging from 0 to 10‰ diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in δ13Cres was negligible in all investigated species. Further, rapid post-illumination changes in δ13Cres (30 min), which increased from 2.6‰ to 5‰ over the course of the day, were examined by positional 13C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in δ13Cres. However, a Rayleigh fractionation of 12C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in δ13Cres. These results are highly relevant since δ13Cres is used in large-scale carbon cycle studies. Copyright © 2009 John Wiley & Sons, Ltd.